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In modern society, sedentary lifestyles prevail on active living, thus perturbing mental and physical health (39). Indeed, it is well documented that exercise improves cognitive functions (39), attenuates the increase in stress hormones (5), and leads to positive emotional effects (11), overall reducing the incidence and severity of stress-related psychiatric disorders, including depression and anxiety (36). Physical activity also improves body energy homeostasis by ameliorating insulin resistance and glucose metabolism and by improving blood lipid profile, that is, by decreasing plasma triglycerides and low-density lipoproteins and by increasing high-density lipoprotein cholesterol (18). Therefore, it is not surprising that regular physical activity has been reported to reduce, in the long term, the risk of obesity, cardiovascular diseases, type 2 diabetes, and metabolic syndrome as well as to ameliorate symptoms of conditions usually characterized by chronic low-grade systemic inflammation (30). Conversely, sedentary lifestyle leads to visceral fat accumulation, paralleled by macrophage infiltration in the adipose tissue, which in turn releases pro-inflammatory cytokines like tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) (28).

In the last decade, a growing body of evidence has demonstrated that exercise of various types, durations, and intensities affects cell immune functions, ultimately altering the balance between pro- and anti-inflammatory cytokines (30). In response to contraction, skeletal muscle also releases several cytokines and peptides (called “myokines”), able to modulate energy metabolism and immune response (30). In this scenario, a key role is played by IL-6, a pleiotropic cytokine that is released from the contracting skeletal muscle depending on intensity and duration of exercise (30). Despite its classification as a pro-inflammatory cytokine, IL-6 has emerged as a key player that accounts for the protective and anti-inflammatory effects of regular exercise (30,39). Indeed, during exercise, IL-6 promotes the release of IL-10 and IL-1 receptor antagonists (both anti-inflammatory cytokines) as well as of the soluble TNF-α receptor, which in turn inhibits TNF-α production (30). In addition, IL-6 enhances insulin sensitivity, hepatic glucose production, lipolysis, and fat oxidation, overall exerting an important metabolic role during exercise (30).

The endocannabinoid system is an ubiquitous and complex signaling network that includes the following: (i) endogenous lipid mediators (collectively termed “endocannabinoids” [eCB]), like N-arachidonoylethanolamine (anandamide [AEA]) and 2-arachidonoylglycerol (2-AG); (ii) their molecular targets, especially type 1 (CB1) and type 2 (CB2) cannabinoid receptors; and (iii) different proteins involved in their synthesis, transport, and degradation (24). Among the latter proteins, a major element is fatty acid amide hydrolase (FAAH), which is crucial in regulating the eCB tone in vivo (9) by degrading AEA, 2-AG, and their congener N-palmitoylethanolamine (PEA) (24).

It has been clearly documented that eCB play a critical role during inflammation and immune response, as they modulate the activation and migration of immune cells (19,34) as well as the expression of inflammatory cytokines, including TNF-α, IL-6, IL-2, IL-8, IL-17, and IL-4 (7,34). Also, the ability of eCB to control energy homeostasis has received a great deal of attention, particularly in the light of their ability to influence appetite and food intake as well as glucose and lipid metabolism (24). It is well known that the eCB system is involved in the onset of obesity and related complications. For instance, AEA inhibits synthesis and release of adiponectin, an insulin-sensitizing and anti-inflammatory cytokine, while it increases synthesis and release of the pro-inflammatory adipocytokine visfatin, whose expression is regulated by insulin resistance-inducing hormones (24). Moreover, CB1 antagonism decreases TNF-α secretion and restores adiponectin release from lipopolysaccharide-treated adipocytes (26).

The available experimental evidence indicates that eCB might account for some of the biological effects triggered by physical exercise. For instance, increased plasma levels of AEA and PEA, but not of 2-AG, have been observed in human subjects after aerobic exercise (11,17,32, 33,37). The increase of AEA levels depends on exercise intensity and occurs upon moderate exercise, but not at low or high exercise intensities (33). Interestingly, the effect of exercise on eCB levels seems to be strictly species specific, with a role for eCB only in those mammals that are morphologically adapted to endurance exercise (32). In this context, it has been proposed that once released into the bloodstream, eCB readily pass through the blood–brain barrier and influence the neurobiological reward associated with exercise, thus contributing to the immediate feeling of well-being (11). In addition, the evidence that eCB exert different effects at central and peripheral levels, and that CB1 and CB2 receptors are localized in muscle, adipose tissue, endothelial cells, and lung (24), suggests a role for eCB in regulating physiological responses to exercise.

However, data on the effects of physical activity on the eCB system are still sparse and sometimes conflicting, possibly because of differences in training protocols (acute vs chronic exercise) and/or in species (humans, mice, rats, or ferrets) used as paradigms. For instance, although short exercise protocols up-regulate eCB transmission in the striatum of mice (10), prolonged aerobic exercise significantly reduces striatal and hippocampal CB1 expression in adolescent rats (14) as well as in adipose tissue of a rat model of metabolic syndrome (43).

Against this background, here we sought to investigate how lifestyle could influence the eCB system. Unlike other studies aimed at analyzing changes in eCB levels in response to acute exercise, our purpose was to study how two different lifestyles (sedentary vs physically active) may influence plasma levels of IL-6 and the key eCB-metabolizing enzyme FAAH, which is known to play a primary role in controlling eCB tone in vivo (9). We demonstrated that, when compared with the sedentary group, active subjects practicing regular aerobic exercise have a significantly higher content of circulating IL-6 as well as increased lymphocyte FAAH activity. We also found an IL-6-mediated enhancement of FAAH activity and expression in vitro, triggered by a cAMP response element (CRE)–like-dependent up-regulation of FAAH promoter activity.

In conclusion, our data demonstrate that IL-6, the most abundant cytokine produced during exercise, can directly regulate FAAH expression and activity, and hence eCB signaling, upon regular exercise.

Fitness evaluation.

Before inclusion in the study, all subjects performed two tests to confirm the differences in aerobic fitness level between groups: the Harvard Step Test (HST), determining the fitness index score (35), and the measurement of maximal oxygen uptake (V˙O2max) through cardiopulmonary maximal stress test at increasing loads on a cycloergometer (Ergocard II, ESAOTE BIOMEDICA®) linked to an ergospirometer (Cardio2 MEDICAL GRAPHICS®). At the beginning of the maximal stress test, the exercise load was fixed at 30 W for 2 min, then it was increased 10 W every min until reaching 85% of maximal heart rate (HRmax) for the age (calculated through the formula: HRmax= 220 − age in years). Blood pressure was assessed through a mercury sphygmomanometer, during and at the end of the test. Subjects were monitored during the recovering period after the effort, for 5 min, through electrocardiogram and blood pressure measurement. According to the literature (12), the test was stopped when one of these situations occurred: symptoms onset, complicated arrhythmias onset, significant ST segment anomalies, muscular exhaustion, and reaching of V˙O2max. According to the classical definition of V˙O2max, we considered that it was reached when: the subject reached a plateau, that is, when V˙O2 increased less than 150 mL·min−1, rising from a step to the next one; respiratory exchange quotient exceeded 1.08–1.10; the subject had more than 10 beats over maximum HR for the age. No subjects showed cardiovascular or respiratory problems, and none had any evidence of musculoskeletal injury.

Sample preparation.

After performing maximal stress test, subjects were instructed not to change their level of physical activity in everyday life nor their dietary habits. Blood samples were collected 1 wk later. No breakfast or coffee was allowed in the morning of blood collection. Active and sedentary subjects provided antecubital venous blood (20 mL per donor) at 8:00 a.m. under resting conditions (i.e., without performing any exercise 12 h before testing). Part of blood was immediately centrifuged at 3000g for 10 min at 4°C to obtain plasma and then kept frozen at −80°C until analysis. Peripheral lymphocytes were purified by gradient centrifugation, using the density separation medium Lympholyte-H, as previously reported (22). After this procedure, isolated cells consisted of lymphocytes (∼85%), B cells (∼10%), and monocytes (∼5%). Cells were resuspended in DMEM-F12 1:1 medium, 2 mM glutamine, 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin, and 10% heat-inactivated fetal bovine serum, at a density of 1.5 × 106 cells per milliliter. The treatment of lymphocytes with IL-6 was performed at 37°C in humidified 5% CO2 atmosphere, at the indicated concentrations and for the indicated periods of time. Controls were incubated with vehicles alone. After each treatment, cell viability was tested by trypan blue dye exclusion and was found to be always ≥95% (22).

Assay of FAAH activity.

The enzymatic activity of FAAH (E.C.3.5.1.4) was assayed in lymphocyte homogenates (50 μg of protein per test) or in whole blood (15 μL per test), by incubating samples with 10 μM [14C]AEA at 37°C for 30 min, in 50 mM Tris–HCl, pH 7.4 (final volume of 500 μL). The reaction was stopped with a mixture of 1 mL chloroform/methanol (1:1, v/v). After centrifugation at 3000g for 5 min, the release of [14C]ethanolamine from [14C]AEA in the aqueous phase was measured in a β-counter (Perkin Elmer Life Science, Boston, MA). FAAH activity was expressed as pmol of [14C]ethanolamine released per minute per milligram of protein or per milliliter of whole blood.

Analysis of FAAH expression.

FAAH protein content was quantified by Western blotting. Lymphocytes were lysed in cold 50 mM Tris–HCl, pH 7.4, containing the protease inhibitors cocktail. Cell lysates (40 μg of protein/lane) were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gels and blotted onto polyvinylidene fluoride membranes. The latter were blocked with 5% nonfat dried milk for 1 h and then incubated with primary anti-FAAH (1:1000) or anti-β-actin (1:30,000) antibodies. After washing and incubating with secondary antibodies (1:3000), detection was carried out using ECL. The semiquantification of the specific immunoreactive bands was performed with Image J software developed by National Institutes of Health (Bethesda, MD).

The detection of FAAH mRNA was performed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Total RNA was extracted from lymphocytes (3 × 106 cells per sample) using TRIzol® reagent, following the manufacturer’s instructions. One microgram of RNA was used for reverse transcription reaction (InPromII kit; Promega, Madison, WI), by prewarming total RNA and random hexamers (1.25 ng·μL−1) at 70°C for 5 min and then by adding ImProm-II buffer, 5 mM MgCl2, 0.5 mM dNTP, 2 U·μL−1 RNaseOUT, 1 μL Improm II Reverse Transcriptase, and diethyl pyrocarbonate-treated water (final volume 20 μL). The reaction was performed using the following RT-PCR program: 25°C per 5 min, 42°C per 1 h, 70°C per 15 min. The target transcripts were amplified using an ABI PRISM 7700 sequence detector system (Applied Biosystems, Foster City, CA), using the following primers: human FAAH Fw (5′-GGTGCAGTACGAGCTGTGGGC-3′) and Rv (5′-GAAGCAGCAGGCCAGGGCG-3′); human β-actin Fw (5′-TGACCCAGATCATGTTTGAG-3′) and Rv (5′-TTAATGTCACGCACGATTTCC-3′). One microliter of cDNA was used for amplification in a 25-μL reaction solution, containing 12.5 μL of Platinum®SYBR®Green qPCRSuper-Mix-UDG (Invitrogen) and 10 pmol of each primer; samples were tested in triplicate. The following PCR program was used: 95°C for 10 min; 40 amplification cycles at 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s. All data were normalized to the endogenous reference gene, β-actin. Differences in quantification cycle (Cq) number were used to calculate the relative amount of PCR target contained in each tube. The relative expression of different gene transcripts was calculated by the delta-delta Cq (ΔΔCq) method and was converted to relative expression ratio (2−ΔΔCq) for statistical analysis. After PCR, a dissociation melting curve was constructed in the 60°C–95°C range to evaluate specificity of the amplification products.

The construction of the vector containing the wild-type (wt) and mutated FAAH promoter region from +1 to –107 nucleotides has already been described (23). The same strategy was used to produce the vector with the +1 to –33 region of the FAAH promoter. Human lymphocytes were transfected, and chloramphenicol acetyltransferase activity was assayed as reported (23).

Quantification of AEA, 2-AG, and PEA plasma levels.

An ice-cold solution of acetone (1 mL), containing 10 pmol of d8-AEA, 500 pmol of d8-2-AG, and 100 pmol of d4-PEA as internal standards, was added to each plasma sample (500 μL). The precipitated proteins were removed by centrifugation (350g for 5 min) and the clear supernatant was recovered and evaporated to dryness. A mixture of methanol–chloroform–water (1:2:1, v/v/v) was added to each sample and centrifuged at 4°C (350g for 5 min). The organic phase was dried and, then, analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS), using a single quadrupole API-150 EX mass spectrometer (Applied Biosystem, CA) in conjunction with a Perkin Elmer LC system (Perkin Elmer, MA). Quantitative analysis was performed by selected ion recording over the respective sodiated molecular ions.

Statistical analysis.

All values are expressed as mean ± SEM of at least three independent experiments. The Student unpaired t-test or one-way ANOVA (followed by Bonferroni Post hoc analysis) was used to compare quantitative data with normal distributions and equal variance. The statistical program Statistical Package for the Social Sciences (version 15.0 for Windows; SPSS Inc., Chicago, IL) was used to perform the analysis, and values of P < 0.05 were considered statistically significant.

RESULTS

Physically active subjects have higher fitness levels.

To assess the influence of regular physical activity on the key eCB-metabolizing enzyme FAAH, sixteen healthy men were enrolled and grouped in physically active and sedentary subjects, on the basis of their fitness level. The physical characteristics of people enrolled in this study are summarized in Table 1. We found that the physically active group had a higher fitness level than the sedentary group (Table 1). Active subjects showed significantly higher V˙O2max (P < 0.05 vs sedentary subjects) and time of exhaustion at maximal exercise values (P < 0.01), as well as a better fitness index score after HST (P < 0.001).

We first ascertained whether plasma IL-6 levels were affected by lifestyle and quantified cytokine concentration in both physically active and sedentary subjects by ELISA. We found that plasma IL-6 concentrations were significantly different in the two groups: circulating IL-6 levels were approximately eightfold higher (P < 0.01) in active than that in sedentary subjects (Fig. 1A). Then, we investigated the effect of an active lifestyle on lymphocyte FAAH activity. Under resting conditions, lymphocyte FAAH activity was approximately fivefold higher (P < 0.001) in subjects who habitually practiced aerobic sport compared with in those who did not (Fig. 1B).

Unlike lymphocyte FAAH activity, whole-blood FAAH activity was not affected by exercise (Table 2). Consistently, we failed to detect any change in plasma levels of the FAAH substrates AEA, 2-AG, and PEA, which remained the same in active and sedentary subjects (Table 2).

IL-6 stimulates FAAH expression and activity in human peripheral lymphocytes in vitro. To further investigate whether lymphocyte FAAH activity could be modulated by circulating IL-6, different concentrations of this cytokine (from 0.1 pg·mL−1 to 100 ng·mL−1) were tested in vitro on FAAH activity of lymphocytes isolated from sedentary subjects. At lower concentrations (up to 1 ng·mL−1), IL-6 failed to increase FAAH activity, which almost doubled (P < 0.01 vs controls) at 10 ng·mL−1 of the cytokine (Fig. 2A). The latter concentration was chosen to perform additional time-course experiments. Incidentally, such a dose of IL-6 is within the range of concentrations commonly used in in vitro studies (6,8). Lymphocytes incubated for up to 24 h with 10 ng·mL−1 IL-6 did not show any significant change in FAAH activity compared with controls, yet treatment for additional 24 h almost doubled (P < 0.01 vs controls) enzyme activity (Fig. 2B).

IL-6 up-regulates FAAH expression through a CRE-like element in the FAAH promoter.

Next, we sought to gain further insight into the mechanism(s) of the IL-6-mediated stimulation of FAAH. In a previous study (23), we have analyzed the FAAH promoter and have found that, like many promoters lacking a TATA box, it had a proximally positioned SP1 site along with another SP1 site in the reverse orientation, 100 nucleotides upstream (Fig. 4A). The promoter sequence showed a CRE-like sequence (Fig. 4A), which is a transcriptional target of the IL-6 family (23). Therefore, we investigated whether IL-6 could influence FAAH expression through this CRE-like element. To this end, we cloned 33nt and 107nt long fragments of the FAAH proximal promoter before the CAT gene and used these constructs in transient transfection experiments with primary human lymphocytes as recipient cells. We found that the minimal promoter (−33 to +1 construct, Fig. 4B), containing only the proximal SP1 site, was unresponsive to IL-6 (Fig. 4B), whereas the 107-nt-long construct, containing both SP1 sites and the CRE-like element, was up-regulated approximately fivefold over controls by treatment with IL-6 (Fig. 4B). Additional transient transfection experiments showed that the FAAH promoter with a mutated CRE-like sequence lost responsiveness to IL-6 (Fig. 4B), thus confirming that the CRE-like site was indeed crucial for the modulation of FAAH expression by the myokine.

DISCUSSION

In the present study, we demonstrated that FAAH, the major eCB-degrading enzyme, can be modulated by lifestyle. By comparing active versus sedentary subjects, we showed that lymphocyte FAAH activity increased in the active group through a mechanism involving IL-6. The modulation of FAAH occurs at the transcriptional level, as demonstrated by transient transfection experiments, where the myokine was shown to up-regulate a CRE-like site in the FAAH promoter; this site appears to be necessary, as its mutation abolished the effect of IL-6 (Fig. 4B). We have previously shown that also the anorexigenic hormone leptin, which belongs to the same interleukin family as IL-6 (1), stimulates lymphocyte FAAH expression via a leptin receptor-mediated activation of STAT3 signaling, which in turn leads to up-regulation of the same CRE-like site targeted by IL-6 (23,27). Thus, STAT3-dependent signal transduction may be activated also by IL-6, as it is activated by leptin (1,44). In this context, it is noteworthy that leptin negatively modulates hypothalamic levels of AEA (24), and that in trained subjects, basal plasma levels of this hormone are low (41). Therefore, it can be proposed that in physically active individuals IL-6 compensates for a reduced leptin in keeping circulating levels of eCB low.

In line with a previous study (13), here we observed that circulating IL-6 levels were significantly higher in physically active than that in sedentary subjects, even after a resting period (Fig. 1A). Remarkably, increased IL-6 levels paralleled increased FAAH activity (Fig. 1B). The relationship between IL-6 and FAAH activity was further confirmed by in vitro experiments, where lymphocytes isolated from sedentary individuals and treated with 10 ng·mL−1 IL-6 exhibited enhanced FAAH expression and activity (Figs. 2 and 3). In this context, we have previously shown that the anti-inflammatory cytokines IL-4 and IL-10 also stimulate expression and activity of human lymphocyte FAAH, whereas the pro-inflammatory cytokines IL-12 and IFN-γ have an opposite effect (22). It should be recalled that different types of physical activity increase plasma concentrations of IL-10 and IL-4, and that IL-6 further enhances IL-10 levels (30). Therefore, it is likely that multiple factors, released by muscle cells as well as by other cells and tissues, can control lymphocyte FAAH during exercise.

Notably, IL-6 gene is rapidly activated in the working skeletal muscle depending on exercise duration, intensity and endurance capacity, and the subsequent increase in blood IL-6 concentration (up to 100-fold) has been shown to develop an anti-inflammatory environment, by increasing production and release of the anti-inflammatory IL-10 and IL-1 receptor antagonists as well as by inhibiting TNF-α production (30,39). Moreover, IL-6 plays an important metabolic role by increasing insulin sensitivity in skeletal muscle, gluconeogenesis in liver, lipolysis, and fat oxidation in adipose tissue, thus enhancing substrate delivery during exercise (30). Given that up-regulation of eCB tone affects peripheral energy homeostasis in favor of fat accumulation (24), we can speculate that high IL-6 levels and then increased FAAH activity, are needed to avoid an excessive production of eCB upon exercise, hence preventing anabolic processes and favoring catabolism and energy supply. In this context, it should be recalled that indeed the biological actions of eCB in vivo are tightly regulated by a metabolic control, where FAAH has been recognized as a major player (9).

Increased FAAH activity in lymphocytes of resting active subjects was not paralleled by any change of enzyme activity in whole blood (Table 2), speaking in favor of a specific regulation of FAAH in distinct blood cells. For instance, it seems noteworthy that also platelets are a major contributor to FAAH activity in blood (21), yet these anucleated cell fragments cannot be transcriptionally regulated by IL-6. In addition, a selective regulation of FAAH by IL-6 might allow a local modulation of eCB tone in lymphocytes only. Indeed, the lack of effect of physical exercise on whole-blood FAAH activity was paralleled by the lack of effect on plasma concentrations of AEA, 2-AG, or PEA (Table 2), compounds that can be all efficiently hydrolyzed by FAAH (24). Apparently, the latter finding is at variance with previous studies, showing that moderate exercise induces AEA release into the bloodstream (11,17,32,33,37). However, in previously mentioned studies, circulating eCB levels were analyzed within 1 h from the bout of aerobic exercise, whereas in this study, eCB content was quantified at least 12 h after the last aerobic exercise; we sought to investigate, indeed, the potential impact of two different lifestyles (sedentary vs physically active) on the eCB system under resting conditions.

Circulating eCB levels may contribute to the biological effects of exercise in different ways. For instance, it is well known that physical activity increases cortisol, which affects energy homeostasis by maintaining adequate blood glucose levels (42). Increased eCB content upon exercise might take part in regulating the hypothalamic–pituitary–adrenal (HPA) axis and hence circulating glucocorticoids (2). Conversely, glucocorticoids increase eCB levels, and the CB1-dependent suppression of excitatory inputs to hypothalamic neurons quickly shuts down the HPA axis (25). More recently, we documented an interplay between glucocorticoids and eCB also in controlling inflammatory processes (3), overall suggesting a negative feedback that might be relevant also during physical exercise.

The increase of eCB upon exercise may also represent a protective mechanism against the unwanted side effects of exercise, like fatigue and pain. Remarkably, activation of CB1 receptors in brain reward regions, as well as in peripheral nerve fibers, contributes to reduce nociception (15) and to generate neurobiological reward (17), thus promoting the so-called “runners high” (11). Moreover, it is known that moderate activity enhances immune function above sedentary levels, whereas excessive amounts of prolonged, high-intensity exercise may impair it. In this context, eCB are known to regulate the expression of pro-inflammatory cytokines (7), and AEA has been shown to reduce lymphocyte proliferation by markedly inhibiting IL-2 release from activated T-lymphocytes (34). Therefore, enhanced FAAH activity might be instrumental in preventing these negative effect of eCB on immune cells.

Besides pro- and anti-inflammatory cytokines, an unbalance between reactive oxygen (ROS) and nitrogen (RNS) species has been implicated in immune response during exercise (39). Indeed, ROS/RNS produced during physical activity modulate gene expression, muscle power, and modeling/repair of myocytes and protect them against infections (31). However, an excess of ROS/RNS can have opposite effects (31). By activating CB1 receptors, eCB trigger inducible and endothelial nitric oxide synthase (NOS) expression and mitochondrial biogenesis and respiration, thus promoting ROS/RNS generation (20,16,40). Therefore, enhanced FAAH activity might contribute to reduce the negative effect of ROS/RNS on immune functions. Altogether, these mechanisms might play an important role in encouraging the habitual aerobic exercise.

In conclusion, here we show that IL-6 leads to activation of the FAAH promoter in human lymphocytes, thus enhancing FAAH activity that modulates the eCB tone in physically active subjects. It can be speculated that the stimulatory effect of IL-6 on FAAH activity is a metabolic adaptation that allows to cope with increased eCB levels upon habitual physical exercise.

The authors are grateful to Dr. Elisa Bisicchia for her valuable help with the RT-qPCR experiments. This investigation was partially supported by Fondazione TERCAS (grant no. 2009–2012) and by the Ministero dell’Istruzione, dell’Università e della Ricerca (grant no. PRIN 2010–2011) to M.M. M.T. is recipient of a Regione Lazio PhD fellowship (grant no. 00011377/2010–2013). R.T. is recipient of a Regione Lazio fellowship (grant no. H81J10000100003/2011–2013).

The authors declare no conflict of interest. Results of the present study do not constitute endorsement by the American College of Sports Medicine.